Figure 4

The mutation of K645, K646, R648 and R650 dissociates CIN85 from EGFR endosome. (A) EGF endocytosis analysis in eGFP/eGFP-CIN85 mutants transfected COS7 cells. COS7 cells expressing eGFP-tagged CIN85 mutant were incubated with 200 ng/ml Alexa Fluor 647 labeled EGF for 30 min on ice and then shifted to 37°C. At indicated time the cells were fixed and immunostained with anti-EGFR antibody. EGFP-tagged CIN85 mutant was visualized by eGFP fluorescence and the labeled EGF was visualized by the fluorescence of Alexa Fluor 647. A selected area is enlarged 6 times to reveal the details. The arrows point the white dots which are the colocalization of EGF (purple), EGFR (red) and eGFP-CIN85 mutant (green). The bar is 10 μm. (B) Plot of the percentage of EGF-EGFR endosomes with eGFP-CIN85 or mutant association. For each mutant, 10 cells were used to calculate white dots and total EGF-EGFR endosomes, and the percentage was normalized against the average percentage in eGFP-CIN85 expressing cell. Student’s t-test was performed. The P values between eGFP-CIN85 expressing cells and eGFP expressing cells or eGFP-CIN85 mutants expressing cells were ***9.34E-05 (10 min) and ***0.000272 (20 min) for eGFP; ***0.000187 (10 min) and **0.001979 (20 min) for eGFP-ΔCC; ***0.000252 (10 min) and **0.007448 (20 min) for eGFP-16 m; **0.001818 (10 min) and **0.002121 (20 min) for eGFP-A4m; 0.329283 (10 min) and 0.739651 (20 min) for eGFP-B4m.